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Biomedical subjects

M F Roberts

Publications and source records attributed to M F Roberts.

At least 145 records · Page 8Linked to original sources
PubMed1978

Phospholipase A2 contamination of cobra venom factor preparations. Biologic role in complement-dependent in vivo reactions and inactivation with p-bromophenacyl bromide.

Cobra venom factor (CoF), the anticomplementary protein in Naja naja cobra venom, is usually purified by sequential ion exchange and gel filtration chromatography. CoF prepared in this manner contains small but significant quantities of phospholipase A2 activity. This acyl hydrolase activity can be simply and efficiently removed on a large scale by treatment of CoF with p-bromophenacyl bromide (BPB), an irreversible modifier of the histidine residue in the active site of phospholipase A2. BPB treatment does not alter the anticomplementary activity of CoF. In vivo experiments utilizing intratracheal injections of control and BPB-treated CoF, as well as pure phospholipase A2, revealed that contaminating phospholipase A2, and not the anticomplementary protein, was responsible for the observed acute neutrophil-associated lung injury. However, phospholipase A2 had no effect on the hypotensive and thrombocytopenic effects of CoF infected intravenously into rabbits. Depletion of circulating C3-C9 by intraperitoneal injections of CoF was not altered by removal of phospholipase A2 activity with BPB.

Acetophenones↗
PubMed1977

Photoactivated heterobifunctional cross-linking reagents which demonstrate the aggregation state of phospholipase A2.

Two novel heterobifunctional cross-linking reagents, which can be used to attach photoactivatable nitroaryl azides to primary amino groups of proteins, have been synthesized. The two compounds, N-5-azido-2-nitrobenzoyloxy-succinimide and ethyl N-5-azido-2-nitrobenzoylaminoacet-imidate-HCl, as well as ethyl 4-azidobenzimidate-HCl have been attached to lysine residues of cobra venom phospholipase A2 without a loss in enzymatic activity. Subsequent illumination of the modified forms of the enzyme at appropriate wavelengths under conditions in which the native enzyme exists in an aggregated state led to the formation of covalently linked dimers and large aggregates which could be separated by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate.

Elapid Venoms↗
PubMed1977

Chemical modification of the histidine residue in phospholipase A2 (Naja naja naja). A case of half-site reactivity.

Reaction of phospholipase A2 (Naja naja naja) with p-bromophenacyl bromidine leads to almost complete loss of enzymatic activity. The rate of inactivation is pH-dependent with pKa equals 6.9 for the ionizing residue. p-Bromophenacyl bromide modifies 0.5 mol of histidine/mol of enzyme as judged by amino acid analysis and incorporation studies with 14C-labeled reagent. The rate of inactivation is affected by various cations; a saturating concentration of Ca2+ decreases the rate 5-fold, while Mn2+ increases the rate by a factor of 2. Triton X-100, which by itself has little affinity for the enzyme, protects against inactivation, presumably by sequestering p-bromophenacyl bromide into the apolar micellar core. The mixed micelle system of Triton X-100, dipalmitoyl phosphatidylcholine, and Ba2+ offers the best protection, lowering the inactivation rate by at least 50-fold. This suggests an active site role for the histidine residue. Ethoxyformic anhydride also modifies phospholipase A2, by acylation of the two amino groups, a tyrosine, and 0.5 mol of histidine/mol of enzyme without totally inactivating the enzyme. Removal of the ethoxyformyl group from the histidine does not reactivate the enzyme. Thus, modification of 0.5 mol of histidine with this reagent is not responsible for the 85% loss of activity seen. Ethoxyformylated enzyme, with 0.5 mol of acylated histidine/mol of enzyme, can be further inactivated by treatment with p-bromophenacyl bromide. The resulting derivative contains 0.4 mol of the 14C-labeled p-bromophenacyl group. Other modifiable groups do not show this half-residue reactivity. For example, oxidation of phospholipase A2 with N-bromosuccinimide leads to rapid destruction of 1.0 tryptophan residue and 5% residual activity. The results of these chemical modification experiments can be interpreted in terms of a model in which the active species of enzyme interacting with mixed micelles is a dimer (or possibly higher order aggregate). The dimer, though composed of identical subunits, is asymmetric; the histidine of one subunit is accessible to ethoxyformic anhydride, while the other histidine is near a hydrophobic region of the enzyme and is chemically reactive toward p-bromophenacyl bromide.

Animals↗
PubMed1977

Dual role of interfacial phospholipid in phospholipase A2 catalysis.

The results of crosslinking experiments with dimethyl suberimidate and gel filtration binding studies are used to delineate a detailed model for phospholipase A2(phosphatide 2-acyl-hydrolase, EC 3.1.1.4) action in the presence of Ca2+ on mixed micelles of Triton X-100 and phospholipid. Important features of the "dual-phospholipid" model are: (i) the use of the nonionic surfactant as an inert matrix that may influence lipid conformation but does not interact with the enzyme; (ii) the involvement of two lipid molecules in a single cycle of catalysis as an explanation for the "surface dilution" phenomenon; (iii) the requirement of an ordered reaction whereby divalent metal ion binds prior to phospholipid binding; and (iv) the induction by lipid substrate of an asymmetric dimer structure for the enzyme.

Binding Sites↗
PubMed1977

Skin blood flow and sweating changes following exercise training and heat acclimation.

Eight subjects underwent an exercise training program (10 days at 75% VO2max for 1 h/day at 25 degrees C db/13 degrees C wb) and a heat-acclimation program (10 days at 50% VO2max for 1 h/day at 35 degrees C db/32 degrees C wb). The relations of chest sweat rate and of forearm blood flow to internal temperature were determined for each subject at a 25 degrees C ambient temperature before training, between training and acclimation, and following acclimation. Training shifted the vasodilation and sweating thresholds toward lower internal temperatures, and acclimation further lowered these thresholds. All threshold shifts were statistically significant (P less than 0.05). Training and acclimation both appeared to increase the slope of the sweating relation, but these effects were not statistically significant. Changes in the slope of the blood flow relation were small and inconsistent. Since arm blood flow is higher at any given internal temperature after acclimation, the lower blood flow which is reported to accompany heat acclimation must result from the lower body temperatures.

Acclimatization↗
PubMed1976

Evidence from 13C NMR for protonation of carbamyl-P and N-(phosphonacetyl)-L-aspartate in the active site of aspartate transcarbamylase.

Nuclear magnetic resonance has been used to study the binding of [13C]carbamyl-P (90% enriched) to the catalytic subunit of Escherichia coli aspartate transcarbamylase. Upon forming a binary complex, there is a small change in the chemical shift of the carbonyl carbon resonance, 2 Hz upfield at pH 7.0, indicating that the environments of the carbonyl group in the active site and in water are similar. When succinate, an analog of L-aspartate, is added to form a ternary complex, there is a large downfield change in the chemical shift for carbamyl-P, consistent with interaction between the carbonyl group and a proton donor of the enzyme. The change might also be caused by a ring current froma nearby aromatic amino acid residue. From the pH dependence of this downfield change and from the effects of L-aspartate analogs other than succinate, the form of the enzyme involved is proposed to be an isomerized ternary complex, previously observed in temperature jump and proton NMR studies. The downfield change to chemical shift for carbamyl-P bound to the isomerized complex is 17.7 +/- 1.0 Hz. Using this value, the relative ability of other four-carbon dicarboxylic acids to form isomerized ternary complexes with the enzyme and carbamyl-P has been evaluated quantitatively. The 13C peak for the transition state analog N-(phosphonacetyl)-L-aspartate (PALA), 90% enriched specifically at the amide carbonyl group, is shifted 20 Hz downfield of the peak for free PALA upon binding to the catalytic subunit at pH 7.0. In contrast, the peak for [1-13C] phosphonaceatmide shifts upfield by about 6 Hz upon binding. Since PALA induces isomerization of the enzyme and phosphonacetamide does not, these data provide further evidence consistent with protonation of the carbonyl group only upon isomerization. The degrees of protonation is strong acids of the carbonyl groups of PALA, phosphonacetamide and urethan (a model for the labile carbamyl-P) have been determined, as have the chemical shifts for these compounds upon full protonation. From these data it is calculated that the amide carbonyl groups of carbamyl-P and PALA might be protonated to a maximum of about 20% in the isomerized complexes at pH 7.0. The change in conformation of the enzyme-carbamyl-P complex upon binding L-aspartate, previously proposed to aid catalysis by compressing the two substrates together in the active site, may be accompanied by polarization of the C=O bond, making this ordinarily unreactive group a much better electrophile. A keto analog of PALA, 4,5-dicarboxy-2-ketopentyl phosphonate, also binds tightly to the catalytic subunit and induces a very similar conformational change, whereas an alcohol analog, 4,5-dicarboxy-2-hydroxypentyl phosphonate, does not bind tightly, indicating the critical importance of an unhindered carbonyl group with trigonal geometry.

Aspartic Acid↗
PubMed1976

Nocturnal lowering of thresholds for sweating and vasodilation.

Six subjects exercised on a bicycle ergometer at 60-70% of maximal aerobic power in a 25 degrees C ambient. Experiments on each subject were conducted at night (4:00-5:30 A.M.) and in daytime (noon-4:30 P.M.). Chest sweating rate (msw) was measured with resistance hygrometry. Forearm blood flow (BF), with an arm skin temperature of 35.5 +/- 1.2 degrees C (SD), was measured with electrocapacitance plethysmography. Esophageal temperature (Tes) was measured with a thermocouple at the level of the left atrium, and mean skin temperature (Tsk) was calculated from a weighted average of temperatures at three sites. Tes was corrected to a skin temperature of 33 degrees C as follows: T'es = Tes + (Tsk - 33 degrees C)/8. This correction reflects the relative contributions of Tes and Tsk to control of msw:T'es and BF:T'es relations were not consistently changed. In any given subject, thresholds for sweating and vasodilation were shifted about equally. These shifts averaged 0.57 degrees C (range: 0.23-0.93 degrees C)for msw and 0.63 degrees C (range: 0.17-0.98 degrees C) for BF.

Adult↗
PubMed1975

Inactivation of Salmonella phosphoribosylpyrophosphate synthetase by oxidation of a specific sulfhydryl group with potassium permanganate.

Phosphoribosylpyrophosphate synthetase from Salmonella typhimurium contains four cysteine residues per subunit. Three of these react readily with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB), forming an active derivative with kinetic and physical properties similar to the native enzyme, but one reacts only under denaturing conditions. Stoichiometric amounts of KMnO4 inactivate the DTNB-treated enzyme. The loss of activity is correlated with the oxidation of the remaining cysteinyl group to cysteic acid by KMnO4. Amino acid analysis indicates that no other residues are altered. The rate of inactivation of the enzyme is decreased 30-fold by saturatin g concentrations of the substrate ATP. Inorganic phosphate also protects substantially against KMnO4. Titration of the native enzyme with limiting amounts of KMnO4 shows that the sulfhydryl group essential for activity competes effectively with the other sulfhydryl groups for KMnO4. These results suggest that the essential sulfhydryl group is near the active site, and that KMnO4, a phosphate analogue, can act as an active site-directed reagent at the ATP binding site of the enzyme. The KMnO4-oxidized enzyme is more highly aggregated than untreated enzyme and fails to bind ATP appreciably.

Adenosine Diphosphate↗
PubMed1975

Forearm blood flow during body temperature transients produced by leg exercise.

Subjects exercised for 30 min on a bicycle ergometer at 30, 50, and 70% of maximal aerobic power in ambient temperatures of 15, 25, and 35 degrees C and vapor pressures of less than 18 Torr. Exercise was used to vary internal temperature during an experiment, and different ambient temperatures were used to vary skin temperatures independently of internal temperature. Forearm skin temperature was fixed at about 36.5 degrees C. Esophageal temperature (Tes) was measured with a thermocouple at the level of the left atrium, and mean skin temperature (Tsk) was calculated from a weighted mean of thermocouple temperatures at eight skin sites. Forearm blood flow (BF) was measured by electrocapacitance plethysmography. Our data are well accounted for by an equation of the form BF = a1Tes + q2Tsk + b, independent of exercise intensity, although some subjects showed an equivocal vasodilator effect of exercise. The ratios a1/a2 (7.5, 9.6, 11.7) are quite similar to the ratios (8.6, 10.4) of the corresponding coefficients in two recent models of thermoregulatory sweating.

Adult↗
PubMed1975

Thermoregulatory control of finger blood flow.

Three men exercised on a bicycle ergometer at 30, 50, asd 70 per cent of maximal aerobic power in ambient temperatures of 15, 25, and 35 degrees C with water vapor pressure less than 18 Torr. Exercies was used to vary internal temperature during as experiment, and different ambient temperatures were used to vary skin temperatures independently of internal temperature. Finger temperature was fixed at about 35.7 degrees C. Espohageal temperature (Tes) was measured with a thermocouple at the level of the left atrium, and mean skin temperature (Tsk) was calcualted from a weighted mean of thermocouple temperatures at eight skin sites. Finger blood flow (BF) was measured by electrocapacitance plethysmography. Although some subjects showed small and equivocal vasomotor effects of exercise, our data are well accounted for by an equation of the form BF equal to alTes + a2Tsk + b, independent of exercise intensity. For these subjects, the ratios a1/a2 (5.9, 8.6, 9.4) were similar to the ratios of the corresponding coefficients recently reported for thermaoregulatory sweating (8.6, 10.4) and for forearm blood flow (9.6).

Adult↗